1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid belonging to the glutamate family using a coryneform bacterium which has been modified so that expression of one or more gene(s) of the NCgl—2067-NCgl—2065 operon in the bacterium is/are attenuated.
2. Brief Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including by transforming microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or U.S. Pat. Nos. 4,346,170; 5,661,012 and 6,040,160).
Many methods for producing L-glutamine by culturing a coryneform bacterium have been disclosed. For example, EP1229121 A2 discloses a method for producing L-glutamine by culturing a coryneform bacterium which has L-glutamine producing ability and has been modified so that its intracellular glutamine synthetase activity is enhanced, and preferably has been further modified so that its intracellular glutamate dehydrogenase activity is enhanced, in a medium to produce and cause accumulation of L-glutamine in the medium, and collecting the L-glutamine.
It is known that transcription of the Bacillus subtilis gene encoding glutamine synthetase (glnA) is regulated by the nitrogen source. The glnA gene is located in an operon with the glnR gene. The glnR gene codes for a negative regulator which directly controls expression of the glutamine synthetase gene (glnA). Certain mutations in glnR gene, such as a large, in-frame deletion and a start codon mutation, lead to high-level constitutivity of the operon; whereas other mutations caused low-level constitutivity (Schreier H J et al., J Mol. Biol. 1989, 210(1): 51-63).
Recently, regulation of glutamine and glutamate metabolism by the nitrogen regulatory protein GlnR in Streptococcus pneumoniae was analyzed. Using DNA microarray analyses of S. pneumoniae D39 wild-type and its isogenic glnR mutant which had been grown in nitrogen-rich medium supplemented with glutamine, a list of operons and genes that were up-regulated the most in the glnR mutant was determined, and includes the following genes: glnA (encoding glutamine synthetase GlnA), glnPQ (encoding ABC transporter amino acid-binding protein/permease), and gdhA (encoding glutamate dehydrogenase glutamate dehydrogenase). All of these genes have a GlnR operator in their promoter regions. Also the zwf gene, encoding the glucose-6-phosphate dehydrogenase and located downstream of, and in the same orientation as glnPQ, was up-regulated (Kloosterman et al., J Biol. Chem. 2006, 281(35):25097-25109). Currently, there have been no reports about the presence of the putative glnR gene in the genomes of coryneform bacteria.
There have been no reports of attenuating expression of one or more genes of the NCgl—2067-NCgl—2065 operon for the purpose of improving productivity of L-amino acid belonging to the glutamate family.